The long-term goals of this project are to describe in detail the macromolecular interactions that take place in the assembly of an active transcription complex on a eukaryotic gene. This will include thermodynamic, kinetic, and structural descriptions of the reactions and molecules involved. Studies proposed here address the transcription of the 5S rRNA genes of Xenopus and are focused on the role played by Transcription Factor IIIA (TFIIIA) both in recognition of the primary transcriptional control region of the 5S rRNA gene and in interactions with other more poorly described protein components of the transcriptional apparatus used by RNA polymerase III. A potential regulatory loop that could play a role in coordinating the production of various components of the ribosome will also be studied in Xenopus embryos. A diverse set of techniques will be used to address these issues, including novel genetic methods we are devising, biochemical analyses of protein-DNA and protein-protein interactions in vitro, phylogenetic comparisons of TFIIIA from evolutionarily distant species, and in vivo studies in developing Xenopus embryos. Results should illuminate the basic biochemical mechanisms underlying the transcription of eukaryotic genes.